IJAMBR 6 (2018) 1-7 ISSN 2053-1818

Recovery of Ophidascaris sp. and Polydelphis sp. from the Indian python in Aizawl district of Mizoram, India

Gautam Patra1*, Kim Jamoh2, Subhamoy Ghosh1, Basanta Saikia3, C. Lalnunpuia1, Apurba Debbarma4, Ajit Kumar4 and Madhurendra Bachan4 , Seikh Sahanawaz Alam5

and Jayashre Bhagawati6

1Department of Veterinary Parasitology, College of Veterinary Sciences and Animal Husbandry,

Central Agricultural University, Selesih, Aizawl, India. 2Department of Veterinary Pathology, College of Veterinary Sciences and Animal Husbandry,

Central Agricultural University, Selesih, Aizawl, India. 3Department of Veterinary Surgery and Radiology, College of Veterinary Sciences and Animal Husbandry,

Central Agricultural University, Selesih, Aizawl, India. 4Department of Veterinary Parasitology, West Bengal University of Animal and Fishery Sciences,

37-K. B. Sarani, Kollkata-700037. 5District Microbiologist, Malda Medical College, Malda, West Bengal, India

6FCLA, College of Veterinary Sciences and Animal Husbandry, Central Agricultural University,

Selesih, Aizawl, India.

Article History ABSTRACT Received 13 November, 2017 Received in revised form 05 December, 2017 Accepted 14 December, 2017 Keywords: Ophidascaris sp., Polydelphis sp., Python, India. Article Type: Full Length Research Article

This paper underscores detailed morphological characteristics of Ophidascaris sp. and Polydelphis sp. recovered after post-mortem from two dead pythons. Ophidascaris sp. and Polydelphis sp. were recovered from the intestine of two dead pythons and subsequently processed for light microscopy (LM) as well as scanning electron microscopy (SEM) study. Based on light microscopical study the collected specimens were confirmed as Ophidascaris sp. and Polydelphis sp. In addition, SEM study showed some morphological structures which are not clear under LM. To the authors’ knowledge this is the first report of Ophidascaris sp. and Polydelphis sp. from python in this part of India.

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INTRODUCTION Parasitic infections are very common in reptiles including snakes throughout the world and several species under the family Ascarididae including Ophidascaris and Polydelphis have been reported by previous researchers (Murvanidze et al., 2015; Ganjali et al., 2015; Sprent, 1969; Biswas et al., 2014). The Asian python belonging to the genus of Python molurus has two sub-divisions:

*Corresponding author. E-mail: dr.gautampatra@yahoo.co.in. Tel: +918582859415.

the Indian python – Python molurus molurus and the Burmese python – Python molurus bivittalis. The light coloured Indian python is found in India, Pakistan, Sri Lanka and Nepal (Keogh et al., 2001; Auliya et al., 2002). Keeping of snakes as pets are rife and they are also looked as popular zoological and private collection. There has been report that Ophidascaris and Polydelphis are merely found in snakes (Taylor et al., 2016). Ophidascaris sp. are commonly occurring nematode in pythonoid and colubrid snakes according to earlier report (Beck and Pantchev, 2006).

Morphologically, the genus Ophidascaris is characterized by almost squared lips, absence of cervical alae, smaller dorsal lips than ventro lateral lips with well developed interlabia (Taylor et al., 2016). Male with large number of the pre- and post-cloacal papillae and bear two sub-equal spicules. Genital organs open near the posterior extremity of the body in both sexes. Polydelphis can be recognized by the absence of cervical alae, hexagonal lips, and absence of interlabia and interlabial glands with sub-equal spicules and caudal papillae of male as in Ophidascaris (Taylor et al., 2016). The aim of the present study was to report as well as to highlight some morphological features of Ophidascaris and Polydelphis studies under light microscope (LM) and scanning electron microscope (SEM). MATERIALS AND METHODS Collection of sample Two Indian pythons (P. molurus molurus) were found dead in a local zoo of Aizawl, Mizoram, India and the same were brought to the Department of Veterinary Parasitology for post mortem examination. Both snakes were carefully examined for the presence of any ecto or endo parasites. On examination a total of 12 round worms of two different species were recovered. The parasites were washed several times in normal physiological saline (0.85%) with the help of camel brush and subsequently transferred to 70% alcohol for further identification. Morphological study All the worms were first observed under stereo zoom microscope (Euromex, Holland) and separated according to sex. One male and one female of each species were put into lactophenol for LM study. Initial morphological study revealed that ten of the parasites belongs to the genus Ophidascaris and two belong to Polydelphis as described by previous reports (Biswas et al., 2014). Preparation of sample for SEM To get the surface morphology SEM was performed. Species from both genera were fixed in 3% glutaraldehyde. Any extracellular debris like mucus, blood or other body fluids and tissue fragments which otherwise hinder the surface to be examined were removed carefully by washing several times in distilled water with a fine camel brush. The samples were fixed at 4°C for 24 h. The fixed parasites were washed in phosphate buffer saline (pH – 7.2) three times and then in double distilled

Int. J. Appl. Microbiol. Biotechnol. Res. 2 water followed by acetone dehydration. After acetone dehydration, the specimens were dried with liquid carbon dioxide at its critical point, that is, 31.5°C at 1100 psi. The specimens are then immersed in tetramethyl saline (TMS) for 5-10 min for two changes at 4°C and then keep at room temperature (25-27°C) to dry. The samples are mounted on aluminium stubs. They were then gold coated in a sputter coat and were examined under SEM [CJSM – 6360 (JEOL)] at the North-eastern Hill University (NEHU), Shillong, Meghalaya in Sophisticated Analytical Instrument Facilities (SAIF) laboratory. RESULTS AND DISCUSSION A total numbers of 10 Ophidascaris sp. and two of Polydelphis sp. were found during post-mortem examination. Parasites recovered during post-mortem examination were identified on the basis of typical morphological features (Bursey and Brooks, 2011). The average length of Ophidascaris varied from 5-8 cm whereas the average length of Polydelphis was 6 cm. The body of Ophidascaris is light brown in colour, dark brown at the head and tail. They are cylindrical in shape. Unfortunately we could not find any male Ophidascaris. Polydelphis were found only on one dead python. Figures 1 and 2 show the gross structure of Ophidascaris while Figure 3 shows the gross structure of Polydelphis sp. SEM study of Ophidascaris revealed longitudinal groove on the mid surface of the body (Figure 4). Body surface also shows lateral and marginal grooves which are not visible under LM (Figure 5). Under SEM the lips of the Ophidascaris sp. are well visible with papillae and each lip is well separated by grooves (Figure 6). Body surface of Polydelphis shows several longitudinal ridges like Ophidascaris (Figure 7). A prominent cervical groove is also visible (Figure 8). The posterior end of male Polydelphis exhibits some irregular corrugated ridges, papillae and two spicules protruding through the cloacal opening (Figure 9). The posterior end of female Polydelphis sp. ends bluntly and shows some irregular ridges and grooves (Figure 10). Somatic papillae could also be observed on the body surface of Ophidascaris and Polydelphis spp. under SEM (Figures 11 and 12).

It is customary to consider three main facets while evaluating parasitism in reptiles: First, the effect and importance of the parasites on the hosts and importance of the parasites on the hosts themselves; second, any possibility of transmission of parasite to domestic animals; and third, the relationship to public health. Ascaridoid nematodes are very commonly found in pythons. Adults embed in the gastric mucosa while larvae migrate through various visceral organs, causing mechanical damage. Sometimes roundworm infestations are the cause of death of snakes, especially pythons. They also cause inflammation of the gut wall, intestinal

Patra et al. 3

Figure 1. Gross structure of Ophidascaris sp.

Figure 2. Ophidascaris sp. in situ.

Figure 3. Gross structure of Polydelphis sp.

Int. J. Appl. Microbiol. Biotechnol. Res. 4

Figure 4. Longitudinal groove of Ophidascaris sp. under SEM.

Figure 5. Lateral and marginal grooves of Ophidascaris sp. under SEM.

Figure 6. Mouth part of Ophidascaris sp. under SEM.

Patra et al. 5

Figure 7. Longitudinal rides on the body of Polydelphis sp. under SEM.

Figure 8. Cervical groove of surface of Polydelphis sp. under SEM.

Figure 9. Posterior end of male Polydelphis sp. under SEM.

Int. J. Appl. Microbiol. Biotechnol. Res. 6

Figure 10. Posterior end of female Polydelphis sp under SEM.

Figure 11. Somatic papillae of Ophidascaris sp. under SEM.

Figure 12. Somatic papillae of Polydelphis sp. under SEM.

Patra et al. 7 infarction and obstruction of the bile and pancreatic ducts (Kutzer and Grünberg, 1965).

Two species of ascaridoid nematodes belonging to two genera – Ophidascaris and Polydelphis were identified postmortem of two python living in the Aizawl zoo, Mizoram, India. The known ascaridoid nematodes commonly infecting snakes are Ophidascaris spp., Hexametra spp. and Polydelphis spp. (Yamaguti, 1961). Placing of Ophidascaris spp. under Ascaridoid is fiercely debated (Sprent, 1970, 1969). The authors concluded that Ophidascaris is a comparatively new step of evaluation and expansion of this species stretches across Central Africa to Asia, on the one side and towards Madagascar to Australia on the other. New species identification of this species continues (Siqueira et al., 2005). The particular composition of the cuticle of the whole body of nematodes and the ornamentations that they possess in the anterior and posterior extremity are the central attention in the study by SEM. The featured analyzed are the longitudinal or transverse striations, longitudinal ridges, cervical groove inflation as well as spination.

When the cuticle is swollen like blister this condition may be turned inflation which is clearly visible on the body surface of Ophidascaris. Papillae are structural nerve endings some of which have tactile

functions others

are chemo-receptors. According to position they are classified into cephalic, cervical and genital papillae. In the present study cephalic and cloacal papillae of Polydelphis sp. are very clearly observed under SEM. The importance of SEM is in its remarkable ability to provide three dimensional images with high magnifications that allow understanding the spatial relationships among surface structures. It could be an additional tool to separate species that appear morphologically identical when examined under light microscope (Hirschmann, 1983). CONCLUSION Based on the above observation, it can be concluded that snakes can be infected with a variety of helminths and the detail morphological study of snake may help for inter disciplinary cooperation among specialist and on a wider level, improve the wildlife inspection process.

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